Generation of patient-derived pluripotent stem cell-lines and CRISPR modified isogenic controls with mutations in the Parkinson's associated GBA gene.

The GBA gene encodes the lysosomal enzyme glucocerebrosidase (GCase), responsible for the hydrolysis of glucocerebroside to glucose and ceramide. Heterozygous GBA mutations have been associated with the development of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We generated two induced pluripotent stem cell (iPSC) lines from PD patients carrying heterozygous GBA W378G or N370S mutations and subsequently produced isogenic control lines using CRISPR/Cas9 genome editing. The patient-derived iPSCs and isogenic control lines maintained full pluripotency, normal karyotypes, and differentiation capacity. All iPSC lines could be differentiated into dopaminergic neurons, thus providing valuable tools for studying PD pathogenesis.

Generation of homozygous PRKN, PINK1 and double PINK1/PRKN knockout cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing.

Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinson's disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinson's disease.

A streamlined CRISPR workflow to introduce mutations and generate isogenic iPSCs for modeling amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) represents a complex neurodegenerative disorder with significant genetic heterogeneity. To date, both the genetic etiology and the underlying molecular mechanisms driving this disease remain poorly understood, although in recent years several studies have highlighted a number of genetic mutations causative for ALS. With these mutations pointing to potential pathways that may be affected within individuals with ALS, having the ability to generate human neurons and other disease relevant cells containing these mutations becomes even more critical if new therapies are to emerge. Recent developments with the advent of induced pluripotent stem cells (iPSCs) and clustered regularly interspaced short palindromic repeats (CRISPR) gene editing fields gave us the tools to introduce or correct a specific mutation at any site within the genome of an iPSC, and thus model the specific contribution of risk mutations. In this study we describe a rapid and efficient way to either introduce a mutation into a control line, or to correct an allele-specific mutation, generating an isogenic control line from patient-derived iPSCs with a given mutation. The mutations introduced were the G94A (also known as G93A) mutation into SOD1 or H517Q into FUS, and the mutation corrected was a patient iPSC line with I114T mutation in SOD1. A combination of small molecules and growth factors were used to guide a stepwise differentiation of the edited cells into motor neurons in order to demonstrate that disease-relevant cells could be generated for downstream applications. Through a combination of iPSCs and CRISPR editing, the cells generated here will provide fundamental insights into the molecular mechanisms underlying neuron degeneration in ALS.

Microfabricated disk technology: Rapid scale up in midbrain organoid generation.

By providing a three-dimensional in vitro culture system with key features of the substantia nigra region in the brain, 3D neuronal organoids derived from human induced pluripotent stem cells (iPSCs) provide living neuronal tissue resembling the midbrain region of the brain. However, a major limitation of conventional brain organoid culture is that it is often labor-intensive, requiring highly specialized personnel for moderate throughput. Additionally, the methods published for long-term cultures require time-consuming maintenance to generate brain organoids in large numbers. With the increasing need for human midbrain organoids (hMOs) to better understand and model Parkinson's disease (PD) in a dish, there is a need to implement new workflows and methods to both generate and maintain hMOs, while minimizing batch to batch variation. In this study, we developed a method with microfabricated disks to scale up the generation of hMOs. This opens up the possibility to generate larger numbers of hMOs, in a manner that minimizes the amount of labor required, while decreasing variability and maintaining the viability of these hMOs over time. Taken together, producing hMOs in this manner opens up the potential for these to be used to further PD studies.

Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy.

SNCA, the first gene associated with Parkinson's disease, encodes the α-synuclein protein, the predominant component within pathological inclusions termed Lewy bodies. The presence of Lewy bodies is one of the classical hallmarks found in the brain of patients with Parkinson's disease, and Lewy bodies have also been observed in patients with other synucleinopathies. However, the study of α-synuclein pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use three-dimensional midbrain organoids, differentiated from human-induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These human midbrain organoids recapitulate key features of α-synuclein pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication human midbrain organoids express elevated levels of α-synuclein and exhibit an age-dependent increase in α-synuclein aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-synuclein. These phosphorylated α-synuclein aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, human midbrain organoids from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson's disease and provide a powerful system to study the pathogenesis of synucleinopathies.

Auto-qPCR; a python-based web app for automated and reproducible analysis of qPCR data.

Quantifying changes in DNA and RNA levels is essential in numerous molecular biology protocols. Quantitative real time PCR (qPCR) techniques have evolved to become commonplace, however, data analysis includes many time-consuming and cumbersome steps, which can lead to mistakes and misinterpretation of data. To address these bottlenecks, we have developed an open-source Python software to automate processing of result spreadsheets from qPCR machines, employing calculations usually performed manually. Auto-qPCR is a tool that saves time when computing qPCR data, helping to ensure reproducibility of qPCR experiment analyses. Our web-based app ( https://auto-q-pcr.com/ ) is easy to use and does not require programming knowledge or software installation. Using Auto-qPCR, we provide examples of data treatment, display and statistical analyses for four different data processing modes within one program: (1) DNA quantification to identify genomic deletion or duplication events; (2) assessment of gene expression levels using an absolute model, and relative quantification (3) with or (4) without a reference sample. Our open access Auto-qPCR software saves the time of manual data analysis and provides a more systematic workflow, minimizing the risk of errors. Our program constitutes a new tool that can be incorporated into bioinformatic and molecular biology pipelines in clinical and research labs.

A Multistep Workflow to Evaluate Newly Generated iPSCs and Their Ability to Generate Different Cell Types.

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality-control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs. Our workflow tests four benchmarks: cell growth, genomic stability, pluripotency, and the ability to form the three germline layers. We also outline a simple test for assessing cell growth and highlight the need to compare different growth media. Genomic integrity in the human iPSCs is analyzed by G-band karyotyping and a qPCR-based test for the detection of common karyotypic abnormalities. Finally, we confirm that the iPSC lines can differentiate into a given cell type, using a trilineage assay, and later confirm that each iPSC can be differentiated into one cell type of interest, with a focus on the generation of cortical neurons. Taken together, we present a multistep quality-control workflow to evaluate newly generated iPSCs and detail the findings on these lines as they are tested within the workflow.

Applying hiPSCs and Biomaterials Towards an Understanding and Treatment of Traumatic Brain Injury.

Traumatic brain injury (TBI) is the leading cause of disability and mortality in children and young adults and has a profound impact on the socio-economic wellbeing of patients and their families. Initially, brain damage is caused by mechanical stress-induced axonal injury and vascular dysfunction, which can include hemorrhage, blood-brain barrier disruption, and ischemia. Subsequent neuronal degeneration, chronic inflammation, demyelination, oxidative stress, and the spread of excitotoxicity can further aggravate disease pathology. Thus, TBI treatment requires prompt intervention to protect against neuronal and vascular degeneration. Rapid advances in the field of stem cells (SCs) have revolutionized the prospect of repairing brain function following TBI. However, more than that, SCs can contribute substantially to our knowledge of this multifaced pathology. Research, based on human induced pluripotent SCs (hiPSCs) can help decode the molecular pathways of degeneration and recovery of neuronal and glial function, which makes these cells valuable tools for drug screening. Additionally, experimental approaches that include hiPSC-derived engineered tissues (brain organoids and bio-printed constructs) and biomaterials represent a step forward for the field of regenerative medicine since they provide a more suitable microenvironment that enhances cell survival and grafting success. In this review, we highlight the important role of hiPSCs in better understanding the molecular pathways of TBI-related pathology and in developing novel therapeutic approaches, building on where we are at present. We summarize some of the most relevant findings for regenerative therapies using biomaterials and outline key challenges for TBI treatments that remain to be addressed.

Defining the Neural Kinome: Strategies and Opportunities for Small Molecule Drug Discovery to Target Neurodegenerative Diseases.

Kinases are highly tractable drug targets that have reached unparalleled success in fields such as cancer but whose potential has not yet been realized in neuroscience. There are currently 55 approved small molecule kinase-targeting drugs, 48 of which have an anticancer indication. The intrinsic complexity linked to central nervous system (CNS) drug development and a lack of validated targets has hindered progress in developing kinase inhibitors for CNS disorders when compared to other therapeutic areas such as oncology. Identification and/or characterization of new kinases as potential drug targets for neurodegenerative diseases will create opportunities for the development of CNS drugs in the future. The track record of kinase inhibitors in other disease indications supports the idea that with the best targets identified small molecule kinase modulators will become impactful therapeutics for neurodegenerative diseases. This Review highlights the imminent need for new therapeutics to treat the most prevalent neurodegenerative diseases as well as the promise of kinase inhibitors to address this need. With a focus on kinases that remain largely unexplored after decades of dedicated research in the kinase field, we offer specific examples of understudied kinases that are supported by patient-derived data as linked to Alzheimer's disease, Parkinson's disease, and/or amyotrophic lateral sclerosis. Finally, we show literature-reported high-quality inhibitors for several understudied kinases and suggest other kinases that merit additional medicinal chemistry efforts to elucidate their therapeutic potential.

Patient-Derived Stem Cells, Another in vitro Model, or the Missing Link Toward Novel Therapies for Autism Spectrum Disorders?

Autism Spectrum Disorders (ASDs) is a multigenic and multifactorial neurodevelopmental group of disorders diagnosed in early childhood, leading to deficits in social interaction, verbal and non-verbal communication and characterized by restricted and repetitive behaviors and interests. To date, genetic, descriptive and mechanistic aspects of the ASDs have been investigated using mouse models and post-mortem brain tissue. More recently, the technology to generate stem cells from patients' samples has brought a new avenue for modeling ASD through 2D and 3D neuronal models that are derived from a patient's own cells, with the goal of building new therapeutic strategies for treating ASDs. This review analyses how studies performed on mouse models and human samples can complement each other, advancing our current knowledge into the pathophysiology of the ASDs. Regardless of the genetic and phenotypic heterogeneities of ASDs, convergent information regarding the molecular and cellular mechanisms involved in these disorders can be extracted from these models. Thus, considering the complexities of these disorders, patient-derived models have immense potential to elucidate molecular deregulations that contributed to the different autistic phenotypes. Through these direct investigations with the human in vitro models, they offer the potential for opening new therapeutic avenues that can be translated into the clinic.

One Step Into the Future: New iPSC Tools to Advance Research in Parkinson's Disease and Neurological Disorders.

Studying Parkinson's disease (PD) in the laboratory presents many challenges, the main one being the limited availability of human cells and tissue from affected individuals. As PD is characterized by a loss of dopaminergic (DA) neurons in the brain, it is nearly impossible for researchers to access and extract these cells from living patients. Thus, in the past PD research has focused on the use of patients' post-mortem tissues, animal models, or immortalized cell lines to dissect cellular pathways of interest. While these strategies deepened our knowledge of pathological mechanisms in PD, they failed to faithfully capture key mechanisms at play in the human brain. The emergence of induced pluripotent stem cell (iPSC) technology is revolutionizing PD research, as it allows for the differentiation and growth of human DA neurons in vitro, holding immense potential not only for modelling PD, but also for identifying novel therapies. However, to reproduce the complexity of the brain's environment, researchers are recognizing the need to further develop and refine iPSC-based tools. In this review, we provide an overview of different systems now available for the study of PD, with a particular emphasis on the potential and limitations of iPSC as research tools to generate more relevant models of PD pathophysiology and advance the drug discovery process.

A Targeted Bivalent Androgen Receptor Binding Compound for Prostate Cancer Therapy.

The androgen-directed treatment of prostate cancer (PCa) is fraught with the recurrent profile of failed treatment due to drug resistance and must be addressed if we are to provide an effective therapeutic option. The most singular difficulty in the treatment of PCa is the failure to respond to classical androgen withdrawal or androgen blockade therapy, which often develops as the malignancy incurs genetic alterations and gain-of-function somatic mutations in the androgen receptor (AR). Physical cellular damaging therapeutic agents, such as radiation or activatable heat-generating transducers would circumvent classical "anti-functional" biological resistance, but to become ultimately effective would require directed application modalities. To this end, we have developed a novel AR-directed therapeutic agent by creating bivalent androgen hormone-AF-2 compounds that bind with high affinity to AR within cells. Here, we used molecular modeling and synthetic chemistry to create a number of compounds by conjugating 5α-dihydrotestosterone (DHT) to various AF-2 motif sequence peptides, through the use of a glycine and other spacer linkers. Our data indicates these compounds will bind to the AR in vitro and that altering the AF-2 peptide composition of the compound does indeed improve affinity for the AR. We also show that many of these bivalent compounds can readily pass through the plasma membrane and effectively compete against androgens alone.

Structure-guided combination therapy to potently improve the function of mutant CFTRs.

Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces. Although individually these compounds marginally improve ΔF508-CFTR folding efficiency, function and stability, their combinations lead to ~50-100% of wild-type-level correction in immortalized and primary human airway epithelia and in mouse nasal epithelia. Likewise, corrector combinations were effective against rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.

Open Science Meets Stem Cells: A New Drug Discovery Approach for Neurodegenerative Disorders.

Neurodegenerative diseases are a challenge for drug discovery, as the biological mechanisms are complex and poorly understood, with a paucity of models that faithfully recapitulate these disorders. Recent advances in stem cell technology have provided a paradigm shift, providing researchers with tools to generate human induced pluripotent stem cells (iPSCs) from patient cells. With the potential to generate any human cell type, we can now generate human neurons and develop "first-of-their-kind" disease-relevant assays for small molecule screening. Now that the tools are in place, it is imperative that we accelerate discoveries from the bench to the clinic. Using traditional closed-door research systems raises barriers to discovery, by restricting access to cells, data and other research findings. Thus, a new strategy is required, and the Montreal Neurological Institute (MNI) and its partners are piloting an "Open Science" model. One signature initiative will be that the MNI biorepository will curate and disseminate patient samples in a more accessible manner through open transfer agreements. This feeds into the MNI open drug discovery platform, focused on developing industry-standard assays with iPSC-derived neurons. All cell lines, reagents and assay findings developed in this open fashion will be made available to academia and industry. By removing the obstacles many universities and companies face in distributing patient samples and assay results, our goal is to accelerate translational medical research and the development of new therapies for devastating neurodegenerative disorders.

Chaperones rescue the energetic landscape of mutant CFTR at single molecule and in cell.

Molecular chaperones are pivotal in folding and degradation of the cellular proteome but their impact on the conformational dynamics of near-native membrane proteins with disease relevance remains unknown. Here we report the effect of chaperone activity on the functional conformation of the temperature-sensitive mutant cystic fibrosis channel (∆F508-CFTR) at the plasma membrane and after reconstitution into phospholipid bilayer. Thermally induced unfolding at 37 °C and concomitant functional inactivation of ∆F508-CFTR are partially suppressed by constitutive activity of Hsc70 and Hsp90 chaperone/co-chaperone at the plasma membrane and post-endoplasmic reticulum compartments in vivo, and at single-molecule level in vitro, indicated by kinetic and thermodynamic remodeling of the mutant gating energetics toward its wild-type counterpart. Thus, molecular chaperones can contribute to functional maintenance of ∆F508-CFTR by reshaping the conformational energetics of its final fold, a mechanism with implication in the regulation of metastable ABC transporters and other plasma membrane proteins activity in health and diseases.The F508 deletion (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF causing mutation. Here the authors show that cytosolic chaperones shift the F508del channel conformation to the native fold by kinetic and thermodynamic remodelling of the gating energetics towards that of wild-type CTFR.

Real time plasmonic qPCR: how fast is ultra-fast? 30 cycles in 54 seconds.

Polymerase Chain Reaction (PCR) is a critical tool for biological research investigators but recently it also has been making a significant impact in clinical, veterinary and agricultural applications. Plasmonic PCR, which employs the very efficient heat transfer of optically irradiated metallic nanoparticles, is a simple and powerful methodology to drive PCR reactions. The scalability of next generation plasmonic PCR technology will introduce various forms of PCR applications ranging from small footprint portable point of care diagnostic devices to large footprint central laboratory multiplexing devices. In a significant advance, we have introduced a real time plasmonic PCR and explored the ability of ultra-fast cycling compatible with both label-free and fluorescence-based monitoring of amplicon production. Furthermore, plasmonic PCR has been substantially optimized to now deliver a 30 cycle PCR in 54 seconds, with a detectable product. The advances described here will have an immediate impact on the further development of the use of plasmonic PCR playing a critical role in rapid point of care diagnostics.

Characterization of the NPC1L1 gene and proteome from an exceptional responder to ezetimibe.

BACKGROUND: Strategies to reduce LDL-cholesterol involve reductions in cholesterol synthesis or absorption. We identified a familial hypercholesterolemia patient with an exceptional response to the cholesterol absorption inhibitor, ezetimibe. Niemann-Pick C 1-like 1 (NPC1L1) is the molecular target of ezetimibe. METHODS AND RESULTS: Sequencing identified nucleotide changes predicted to change amino acids 52 (L52P), 300 (I300T) and 489 (S489G) in exceptional NPC1L1. In silico analyses identified increased stability and cholesterol binding affinity in L52P-NPC1L1 versus WT-NPC1L1. HEK293 cells overexpressing WT-NPC1L1 or NPC1L1 harboring amino acid changes singly or in combination (Comb-NPC1L1) had reduced cholesterol uptake in Comb-NPC1L1 when ezetimibe was present. Cholesterol uptake was reduced by ezetimibe in L52P-NPC1L1, I300T-NPC1L1, but increased in S489G-NPC1L1 overexpressing cells. Immunolocalization studies found preferential plasma membrane localization of mutant NPC1L1 independent of ezetimibe. Flotillin 1 and 2 expression was reduced and binding to Comb-NPC1L1 was reduced independent of ezetimibe exposure. Proteomic analyses identified increased association with proteins that modulate intermediate filament proteins in Comb-NPC1L1 versus WT-NPC1L1 treated with ezetimibe. CONCLUSION: This is the first detailed analysis of the role of NPC1L1 mutations in an exceptional responder to ezetimibe. The results point to a complex set of events in which the combined mutations were shown to affect cholesterol uptake in the presence of ezetimibe. Proteomic analysis suggests that the exceptional response may also lie in the nature of interactions with cytosolic proteins.

Changing genetic paradigms: creating next-generation genetic databases as tools to understand the emerging complexities of genotype/phenotype relationships.

Understanding genotype/phenotype relationships has become more complicated as increasing amounts of inter- and intra-tissue genetic heterogeneity have been revealed through next-generation sequencing and evidence showing that factors such as epigenetic modifications, non-coding RNAs and RNA editing can play an important role in determining phenotype. Such findings have challenged a number of classic genetic assumptions including (i) analysis of genomic sequence obtained from blood is an accurate reflection of the genotype responsible for phenotype expression in an individual; (ii) that significant genetic alterations will be found only in diseased individuals, in germline tissues in inherited diseases, or in specific diseased tissues in somatic diseases such as cancer; and (iii) that mutation rates in putative disease-associated genes solely determine disease phenotypes. With the breakdown of our traditional understanding of genotype to phenotype relationships, it is becoming increasingly apparent that new analytical tools will be required to determine the relationship between genotype and phenotypic expression. To this end, we are proposing that next-generation genetic database (NGDB) platforms be created that include new bioinformatics tools based on algorithms that can evaluate genetic heterogeneity, as well as powerful systems biology analysis tools to actively process and evaluate the vast amounts of both genomic and genomic-modifying information required to reveal the true relationships between genotype and phenotype.

Cancer systems biology in the genome sequencing era: part 1, dissecting and modeling of tumor clones and their networks.

Recent tumor genome sequencing confirmed that one tumor often consists of multiple cell subpopulations (clones) which bear different, but related, genetic profiles such as mutation and copy number variation profiles. Thus far, one tumor has been viewed as a whole entity in cancer functional studies. With the advances of genome sequencing and computational analysis, we are able to quantify and computationally dissect clones from tumors, and then conduct clone-based analysis. Emerging technologies such as single-cell genome sequencing and RNA-Seq could profile tumor clones. Thus, we should reconsider how to conduct cancer systems biology studies in the genome sequencing era. We will outline new directions for conducting cancer systems biology by considering that genome sequencing technology can be used for dissecting, quantifying and genetically characterizing clones from tumors. Topics discussed in Part 1 of this review include computationally quantifying of tumor subpopulations; clone-based network modeling, cancer hallmark-based networks and their high-order rewiring principles and the principles of cell survival networks of fast-growing clones.

Cancer systems biology in the genome sequencing era: part 2, evolutionary dynamics of tumor clonal networks and drug resistance.

A tumor often consists of multiple cell subpopulations (clones). Current chemo-treatments often target one clone of a tumor. Although the drug kills that clone, other clones overtake it and the tumor recurs. Genome sequencing and computational analysis allows to computational dissection of clones from tumors, while singe-cell genome sequencing including RNA-Seq allows profiling of these clones. This opens a new window for treating a tumor as a system in which clones are evolving. Future cancer systems biology studies should consider a tumor as an evolving system with multiple clones. Therefore, topics discussed in Part 2 of this review include evolutionary dynamics of clonal networks, early-warning signals (e.g., genome duplication events) for formation of fast-growing clones, dissecting tumor heterogeneity, and modeling of clone-clone-stroma interactions for drug resistance. The ultimate goal of the future systems biology analysis is to obtain a 'whole-system' understanding of a tumor and therefore provides a more efficient and personalized management strategies for cancer patients.